GFLV replication in electroporated grapevine protoplasts

Grapevine fanleaf virus (GFLV), responsible for the economically important court-noue disease, is exclusively transmitted to its natural host in the v ineyards through Xiphinema nematodes. We have developed direct inoculation of GFLV into grapevine through protoplast electroporation. Protoplasts were isolated from mesophyll of in vitro-grown plants and from embryogenic cell suspensions. Permeation conditions were determined by monitoring calcein u ptake. Low salt poration medium was selected. Electrical conditions leading to strong transient gene expression were also tested for GFLV inoculation (isolate F13). GFLV replication was detected with either virus particles (2 mu g) or viral RNA (10 ng) in both protoplast populations, as shown by ant i-P38 Western blotting. Direct inoculation and replication were also observ ed with Arabis mosaic virus (ArMV), a closely related nepovirus, as well as with another GFLV isolate. These results will be valuable in grapevine bio technology, for GFLV replication studies, transgenic plant screening for GF LV resistance, and biorisk evaluation. (C) 2000 Elsevier Science ireland Lt d. All rights reserved.